CYP1A2 expression rather than genotype is associated with olanzapine concentration in psychiatric patients

Olanzapine is a commonly prescribed atypical antipsychotic agent for treatment of patients with schizophrenia and bipolar disorders. Previous in vitro studies using human liver microsomes identified CYP1A2 and CYP2D6 enzymes being responsible for CYP-mediated metabolism of olanzapine. The present work focused on the impact of CYP1A2 and CYP2D6 genetic polymorphisms as well as of CYP1A2 metabolizing capacity influenced by non-genetic factors (sex, age, smoking) on olanzapine blood concentration in patients with psychiatric disorders (N = 139). CYP2D6 genotype-based phenotype appeared to have negligible contribution to olanzapine metabolism, whereas a dominant role of CYP1A2 in olanzapine exposure was confirmed. However, CYP1A2 expression rather than CYP1A2 genetic variability was demonstrated to be associated with olanzapine concentration in patients. Significant contribution of − 163C > A (rs762551), the most common SNP (single nucleotide polymorphism) in CYP1A2 gene, to enhanced inducibility was confirmed by an increase in CYP1A2 mRNA expression in smokers carrying − 163A, and smoking was found to have appreciable impact on olanzapine concentration normalized by the dose/bodyweight. Furthermore, patients’ olanzapine exposure was in strong association with CYP1A2 expression; therefore, assaying CYP1A2 mRNA level in leukocytes can be an appropriate tool for the estimation of patients’ olanzapine metabolizing capacity and may be relevant in optimizing olanzapine dosage.

Inclusion criteria were the age of 18 years or older and stable olanzapine therapy for at least two weeks.Exclusion criteria were drug or alcohol addiction.The study was approved by the Hungarian Committee of Science and Ethics, Medical Research Council.It was performed under the regulations of Act CLIV of 1997 on Health and the decree 23/2002 of the Minister of Health of Hungary, and in accordance with the declaration of Helsinki.All patients belonged to the Caucasian population, and their demographic and clinical data as well as the details of olanzapine therapy (daily dose, serum concentration of olanzapine) were recorded (Table 1).
For assaying CYP1A2 mRNA expression, total RNA was extracted from leukocytes (approximately 10 7 cells) isolated from peripheral blood samples using TRIzol reagent (Invitrogen, Carlsbad, CA), according to the manufacturer's instructions.RNA (3 µg) was reverse-transcribed into single-stranded cDNA using Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, CA), and real-time PCR (polymerase chain reaction) with human cDNA was performed using KAPA Fast Probes Mastermix (KAPA Biosystems, Cape Town, South Africa) and TaqMan probe for CYP1A2 (Microsynth AG, Balgach, Switzerland).The quantities of the CYP1A2 mRNA relative to that of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were determined 43 .Patients were classified as low, normal and high CYP1A2 expressers on the basis of CYP1A2 mRNA levels in leukocytes with the cut-off expression values of 10 −5 and 5 * 10 −4 as previously determined 35 .

Plasma concentrations of olanzapine
The blood samples were taken directly before the morning dose of olanzapine, 12 h after the evening dose.The blood samples were taken at the same time for CYPtesting and for therapeutic drug monitoring.Olanzapine plasma concentration was determined by LC-MS/MS using an Inertsil ODS-4 column (75 × 2.1 mm, 3 µm; GL Sciences Inc., Tokyo, Japan) and mobile phases of 0.1% formic acid and acetonitrile in gradient running mode.The samples were analyzed using positive electrospray ionization (Sciex API 2000, MDS AB Sciex, Toronto, Canada) and multiple reaction monitoring mode for quantitation of olanzapine (m/z 313.3/256.1 and 313.3/198.1)..The genetic linkage between CYP1A2 SNPs was demonstrated on the basis of linkage disequilibrium coefficient (D′), correlation coefficient (R 2 ) and logarithm of odds (LOD) calculated by the software Haploview (v.4.2; Broad Institute, Cambridge, MA) 46 .Strong linkage disequilibrium was identified between a pair of SNPs with D′ value more than 0.99 and LOD ≥ 3. Linear regression models were built for identification of potential associations between olanzapine plasma concentration as a dependent variable (experimental results) and the independent variables, such as CYP1A2 and CYP2D6 SNPs, haplotypes, CYP1A2 expression, sex, age (under 50 and over 50 years old) and smoking behaviour (part of experimental setup).Multiple linear regression analyses were carried out by IBM SPSS Statistics software [v28.0.1.0(142), IBM Corp., Armonk, NY, USA].A P value of < 0.05 was considered to be statistically significant.

Data analysis
Statistical significance of CYP1A2 expression, CYP2D6 genotype and smoking behaviour as covariates of olanzapine plasma concentrations was also analyzed by principal component analysis (PCA) and partial leastsquare (PLS) modelling (SIMCA, Sartorius Stedim Data Analytics AB, Umea, Sweden).PCA is a linear method that is efficiently used to decorrelate variables in multivariate data analysis.The method is particularly useful when independent variables cannot be decorrelated upfront via design of experiments (often the case in clinical studies) to extract most from the experimental results and to avoid spurious correlations between independent and dependent variables.PLS provides linear regression models similar to multiple regression; however, it uses PCA to first decorrelate the independent variables of the model.PLS models are identical to multiple regression models when independent variables are uncorrelated and are less biased than multiple regression models when independent variables are correlated.All independent variables of the study were selected as PLS model input variables while predicting olanzapine plasma concentration.The final PLS model was then cleaned of all the independent variables that did not significantly contribute to the model prediction.Cleaning was done on the basis of significance and clinical relevance of each independent variable.Contribution of a variable was considered as significant when estimated corresponding coefficient's absolute value was larger than the estimated standard deviation of the coefficient.In the selection process, centered and scaled model coefficients were used that allowed direct comparison between the coefficients as they were normalized by the corresponding variable's means and standard deviations.Diagonal line and dispersion around it on observed vs model-based predictions of olanzapine plasma concentration plot shows how well the model correlates with measured concentrations.Coefficient of determination (R 2 ) was used as a formal measure of model fit.It provided a measure of how well observed outcomes were predicted by the model.Additionally, coefficient of determination was calculated also for the part of data set not used to estimate the model coefficient and was denoted as Q 2 .Ideally R 2 and Q 2 should be similar for relevant models.
The comparison of normalized olanzapine plasma concentrations between various CYP1A2 or CYP2D6 groups was performed by Kruskal-Wallis ANOVA followed by Dunn's multiple comparisons or Mann-Whitney test (GraphPad Instat v3.05; GraphPad Software, San Diego, CA, USA).

Informed consent
Written informed consent was obtained from all participants.

Patients' genetically determined drug metabolizing capacity and olanzapine exposure
Previous in vitro studies using human liver microsomes and cDNA-expressed microsomal enzymes have indicated that the oxidative metabolism of olanzapine is primarily CYP-mediated with a major role of CYP1A2 and with a minor role of CYP2D6 19 .The association of olanzapine plasma concentration normalized by the dose/ bodyweight (C/D) with CYP1A2 and CYP2D6-status was investigated in patients with psychiatric disorders.No significant differences in olanzapine plasma concentration were observed between various CYP1A2 genotypes reconstructing from the CYP1A2 SNP data of patients (N = 114, P = 0.2444) (Fig. 2A).We have previously demonstrated that CYP1A2 expression rather than CYP1A2 genotype indicated hepatic CYP1A2 activity 35 ; therefore, the association between CYP1A2 mRNA levels in the patients' leukocytes and olanzapine concentration was also established.Due to the low number of high CYP1A2 expressers, in statistical analysis, high and normal expressers were combined.Patients with low CYP1A2 mRNA expression (N = 82) displayed approximately twofold higher olanzapine plasma concentrations than those with normal/high expression (N = 32) [239.2 ± 97.3 (ng/mL)/(mg/ kg) for low versus 120.4 ± 46.8 (ng/mL)/(mg/kg) for normal/high, P < 0.0001] (Fig. 2B).www.nature.com/scientificreports/CYP2D6 genotype has been suggested to be consistently applied for prediction of patients' CYP2D6 metabolic capacity 29 ; therefore, the contribution of patients' phenotype predicted from CYP2D6 genotype to olanzapine plasma concentration normalized by dose/bodyweight was also analyzed.No statistically significant differences in olanzapine concentration were demonstrated between poor, intermediate, normal and ultrarapid CYP2D6 metabolizer subjects [poor: 248.8 ± 39.5 (ng/mL)/(mg/kg), intermediate: 201.0 ± 91.7 (ng/mL)/(mg/kg), normal: 201.6 ± 108.6 (ng/mL)/(mg/kg), ultrarapid: 248.4 ± 136.14 (ng/mL)/(mg/kg); P = 0.2801] (Fig. 2C).

Impact of genetic and non-genetic factors on olanzapine plasma concentration
In addition to the CYP1A2 and CYP2D6 variations, a multiple linear regression analysis was performed to estimate the influence of non-genetic factors, sex and age as well as of tobacco smoking, the CYP1A2 inducing factor on olanzapine exposure in patients with psychiatric disorders (Table 3).Significant association of olanzapine plasma concentration normalized by dose/bodyweight was demonstrated both with tobacco smoking (P = 0.004) and with CYP1A2 expression (P < 0.0001).The olanzapine concentration was approximately 1.5-fold higher in non-smoker patients (N = 64) compared to smokers (N = 46) [non-smokers: 235.9 ± 102.3 (ng/mL)/(mg/kg) vs smokers: 165.6 ± 79.2 (ng/mL)/(mg/kg), P < 0.0001] (Fig. 3A).According to the multivariate analysis, none of the CYP1A2 haplotypes had significant impact on olanzapine exposure in psychiatric patients; however, the contribution of the − 163C > A SNP was assumed to influence CYP1A2 induction by smoking.It was clearly demonstrated that smoking significantly increased CYP1A2 mRNA expression in leukocytes of patients with − 163C/A and − 163A/A nucleotide changes compared to the − 163C/C wild-type patients (C/A: 2.5*10 −5 ± 3.1*10 −5 ; A/A: 4.0 *10 −5 ± 1.2*10 −4 and C/C: 3.2*10 −6 ± 6.8*10 −6 , P = 0.0096); however, the − 163C > A polymorphism did not have an impact on CYP1A2 transcription in non-smoker patients (Fig. 3B,C).Furthermore, the minor role of CYP2D6 in olanzapine metabolism previously demonstrated in human liver microsomes 19 was not confirmed in patients.CYP2D6 genotype-based phenotypes appeared to have no effect on olanzapine plasma concentration.

Dominant factors contributing to olanzapine concentration
In routine clinical practice, olanzapine is administered according to the consensus therapeutic guideline to achieve the optimal therapeutic concentration (20-80 ng/mL) for treatment of schizophrenia and bipolar disorder 14 supplemented with monitoring the relief of symptoms and appearance of side effects.To determine the relative role of key factors influencing olanzapine plasma concentration in psychiatric patients, the PLS model was applied with olanzapine daily dose (mg/kg), CYP1A2 genotype, CYP1A2 expression, CYP2D6 genotypebased phenotype, sex, age and smoking behaviour as input variables and olanzapine plasma concentration as the output variable (Fig. 4A).The input variables without significant contribution to olanzapine concentration regarding the distribution of centered and scaled model coefficients, such as CYP1A2 genotype, sex, age and CYP2D6 genotype-based phenotype were eliminated from model building.The final PLS model equation with the main contributing input factors, such as olanzapine daily dose/bodyweight, CYP1A2 expression and smoking was described as follows: where "cc olanzapine " is olanzapine plasma concentration predicted from the model (ng/mL), "CYP1A2" is − 0.989 for normal/high CYP1A2 expressers or 0.989 for low CYP1A2 expressers, "D" is the daily dose of olanzapine cc olanzapine = (3.187+ CYP1A2 + 12.08 × D + S) 2 (mg/kg), "S" is − 0.273 for smokers or 0.273 for non-smoker patients.The value R 2 (0.64) and Q 2 (0.61) showed a considerable prediction power for the PLS model, suggesting that olanzapine daily dose, patient's CYP1A2 expression phenotype and smoking were mostly responsible for olanzapine plasma concentration variability in psychiatric patients (Fig. 4B).It should be noted that the derived model is non-linear as best prediction was obtained for the squared root of the olanzapine plasma concentration.

Discussion
High interindividual variability in olanzapine pharmacokinetics may impact both the patients' response to the drug and the development of adverse effects which may eventually limit the success of antipsychotic therapy.The present study involving patients with psychiatric disorders investigated the role of CYP1A2 and CYP2D6 genetic variants and patients' CYP1A2 metabolic capacity influenced by non-genetic factors (e.g., sex, age, smoking behaviour) in olanzapine exposure.Although CYP2D6 seems to have minor contribution to olanzapine metabolism and has negligible effect on plasma concentration in vivo 10,17,25,56 , interindividual variability in CYP1A2 activity has been reported to be associated with olanzapine exposure and patients' dose requirement 50 .
In the patients of the present study, the trough concentrations of olanzapine substantially varied between 1.3 and 170 ng/mL (0.0042-0.54 µM), and at high plasma levels, oxidative enzymes other than CYP1A2 may be assumed to be additionally involved in olanzapine metabolism.However, one must also consider that the www.nature.com/scientificreports/Km values (Michaelis constant) for the formation of olanzapine metabolites by various enzymes (14-22 µM for CYP1A2; 638 µM for CYP2D6; 228 µM for FMO3) far exceed the highest plasma concentrations in patients 57 ; therefore, CYP1A2 was likely to be the dominant enzyme in the metabolism.Furthermore, the patients had a wide range of bodyweight from lean (43 kg) to obese (122 kg) that might have had an impact on olanzapine pharmacokinetics.The obesity is known to influence several physiological processes important in ADME (absorption, distribution, metabolism, excretion) parameters of drugs, e.g., increasing gut permeability and liver blood flow, altering drug distribution in tissues and drug metabolizing function of the liver 58 .Therefore, pharmacokinetic properties of a drug may be altered by obesity.Olanzapine is a lipophilic compound (logP is over 3) and may easily diffuse into adipose tissue increasing volume distribution 59 .However, CYP1A2 function has been reported to be unaltered in obese patients 60 , and the rate of olanzapine metabolism is not expected to be modified due to obesity.The variability of CYP1A2 function is linked to genetic polymorphisms resulting in altered enzyme activity; however, haplotype misidentification from SNPs often leads to inconsistent phenotype estimation and allele frequency data in the literature 35,39,[61][62][63] .Some clinical relevance has been attributed to CYP1A2*1F, one of the most frequently studied alleles 27 .Several authors considered CYP1A2*1F to be identical with − 163C > A SNP; however, − 163C > A appears to be in close genetic linkage with − 3860G > A, − 2467delT, − 739T > G or 2159G > A SNPs in CYP1A2*1J, CYP1A2*1K, CYP1A2*1L, CYP1A2*1M, CYP1A2*1V or CYP1A2*1W haplotypes 47,64 .Involving the most common SNPs in CYP1A2 gene into the haplotype reconstruction, CYP1A2*1F was demonstrated to be one of the rarest CYP1A2 alleles in the present patient population with a frequency of 0.4%, in contrast to the 32-57% of literature data 27 .The low prevalence thus did not allow to investigate the effect of CYP1A2*1F on enzyme activity and olanzapine exposure.Nevertheless, the contribution of − 163C > A to CYP1A2 inducibility has been well demonstrated, and several in vivo studies observed elevated CYP1A2 activity in − 163A carrier subjects with smoking or under CYP1A2 inducer omeprazole therapy 42,47,65,66 .In the present study, the smoker patients with − 163C/A or − 163A/A genotypes displayed significantly higher CYP1A2 mRNA expression than the − 163C/C carrier smokers; however, this higher inducibility of − 163A carriers was not manifested in low olanzapine plasma concentrations which was in line with the findings of Czerwensky et al. 23 .Decreased CYP1A2 activity was reported to be associated with − 3860G > A and to − 24667delT 37,38 ; however, no significant association was found between these SNPs and olanzapine plasma concentrations in the patients involved in the present study.These results confirmed the observation of a recent clinical study that CYP1A2 genotype has no significant predictive power for olanzapine exposure 67 .Similarly to CYP1A2, CYP2D6 alleles had no effect on olanzapine exposure in psychiatric patients.
Strong correlation between hepatic CYP1A2 activity and mRNA expression has been demonstrated in previous studies with liver tissue donors 35,68 .Furthermore, CYP1A2 expression in leukocytes has been reported to reflect hepatic CYP1A2 activity; thus, leukocytes are considered to be appropriate biological samples for prediction of patients' CYP1A2 metabolizing capacity 26 .In the patients with psychiatric disorders of the present study, significant association was observed between CYP1A2 expression in leukocytes and olanzapine metabolizing activity.The olanzapine concentration normalized by the dose/bodyweight was approximately twofold higher in the patients with poor CYP1A2 metabolizing capacity compared to intermediate/extensive metabolizer subjects.Several non-genetic intrinsic and environmental factors (e.g., sex, age, nutrition, diseases, hormonal status, smoking and medication) regulating CYP1A2 transcription or inhibiting CYP1A2 enzyme function have www.nature.com/scientificreports/been demonstrated to contribute to the interindividual variability of CYP1A2 phenotype 27,69,70 .Clear association between smoking and increased CYP1A2 activity has been described, and several components of tobacco smoke have been demonstrated to induce CYP1A2 transcription via aromatic hydrocarbon receptor mediated pathway and to increase CYP1A2 metabolic capacity 71 .Furthermore, smoking, the well-studied behaviour seems to prevail within the population of psychiatric patients, primarily of those with schizophrenia 56,72,73 ; therefore, an attention must be paid to the effect of smoking on olanzapine exposure.Approximately 1.5-fold higher olanzapine plasma concentrations were observed in the non-smoker patients of the present study than in smokers, which confirmed the findings of previous studies 17,50,74 .Low olanzapine exposure in smokers was attributed to the smoking induced increase in CYP1A2 expression and activity; therefore, patients' smoking behaviour is suggested to be considered in optimal dose settings for efficient olanzapine therapy in psychiatric patients 75,76 .The PLS model built in the present study also supported the significant role of smoking and CYP1A2 expression as independent variables in olanzapine exposure.Considering these factors together, the model provided a good prediction for olanzapine serum concentrations.Age has been suggested to have negative impact on drug metabolism, generally resulting in lower clearance in elderly patients (> 65) than in younger subjects 77 .An increase in olanzapine concentration by an average of 9.4% per decade of life was observed by Weiss et al. 78 .Sex differences also appear to influence olanzapine pharmacokinetics with reduced clearance in women that has been explained by the evidence for females having lower hepatic CYP1A2 activity 25,74,79 .However, in the present patient population, no statistically significant association was demonstrated between olanzapine exposure and age or sex, most probably due to other non-genetic variables influencing CYP1A2 function that were able to mask the effect of age and sex.Furthermore, it should be noted that only two patients were older than 65 years of age that did not allow to demonstrate a decrease in CYP1A2 function in elderly subjects.
Pharmacokinetic drug-interactions evoking CYP1A2 induction or inhibition of CYP1A2 activity can obviously modify the clearance of CYP1A2 substrate drugs 80,81 .It is highly relevant for psychiatric patients, because they are often under multidrug therapy including antipsychotics, mood stabilizers, antidepressant and sedative agents or under other drugs for treatment of comorbid medical conditions 82 .Co-administration of potent CYP1A2 inhibitor drugs (e.g.ciprofloxacin, fluvoxamine or oral contraceptives) has been reported to significantly elevate the serum concentrations of CYP1A2 substrates, while CYP1A2 inducer drugs, such as carbamazepine and omeprazole, are known to significantly increase the clearance of CYP1A2 substrates, including olanzapine 80,81,83 .In the present study, none of the patients were reported to receive fluvoxamine, carbamazepine or omeprazole, and only one subject displaying low CYP1A2 expression was on ciprofloxacin therapy; however, ciprofloxacin was likely to have negligible impact on the patient's intrinsic poor metabolizer phenotype.No information about oral contraceptive therapy was available in the patients' medication history which was one of the limitations of the present study.

Conclusion
The present study focused on the impact of CYP1A2 and CYP2D6 genetic polymorphisms as well as CYP1A2 metabolizing capacity influenced by non-genetic factors (sex, age, smoking behaviour) on olanzapine blood concentration in patients with psychiatric disorders.CYP2D6 appeared to have negligible contribution to olanzapine metabolism, whereas a dominant role of CYP1A2 in olanzapine exposure was confirmed.However, we first demonstrated that CYP1A2 mRNA expression rather than CYP1A2 genetic variability was associated with olanzapine concentration in patients.Involving the most common SNPs in CYP1A2 gene into the haplotype reconstruction, CYP1A2*1F was found to be one of the rarest CYP1A2 alleles in the present psychiatric population with a frequency of 0.4%, because the − 163C > A (rs762551) SNP designating CYP1A2*1F occurred in genetic linkage with other SNPs (− 3860G > A, − 2467delT, − 739T > G or 2159G > A) in CYP1A2*1L, CYP1A2*1M, CYP1A2*1V or CYP1A2*1W alleles.Significant contribution of − 163C > A to enhanced CYP1A2 inducibility was confirmed by an increase in CYP1A2 mRNA expression in smoker − 163A carriers.Tobacco smoking was proved to be a dominant non-genetic variable that increased olanzapine metabolizing capacity in patients, whereas sex and age appeared to have no impact on olanzapine exposure.Furthermore, it has been clearly demonstrated that patients' olanzapine exposure (C/D) was in strong association with CYP1A2 mRNA expression; thus, assaying CYP1A2 mRNA level in patients' leukocytes can be an appropriate tool for the estimation of their CYP1A2 metabolizing capacity and may facilitate to avoid misdosing induced adverse reactions or olanzapine inefficacy.

Figure 3 .
Figure 3.The influence of patients' smoking behaviour on olanzapine plasma concentrations normalized by the dose/bodyweight (bw) (A) and on CYP1A2 expression of − 163A carrier or non-carrier patients (B and C).*P < 0.001, **P < 0.0001.

Figure 4 .
Figure 4. PLS model for olanzapine plasma concentration.(A) Principal component analysis with input variables of olanzapine daily dose/patients' bodyweight, patients' CYP1A2 expression, smoking behaviour and CYP2D6 genotype-based phenotype; (B) Olanzapine plasma concentrations predicted by the PLS model.

Table 3 .
Multivariate analysis of olanzapine plasma concentration normalized by dose/bodyweight considering genetic (CYP1A2, CYP2D6 haplotypes and phenotypes) and non-genetic factors in psychiatric patients.For CYP1A2 haplotypes, the nucleotid changes are indicated in bold.The P values < 0.05 were considered to be statistically significant and are indicated in bold.